DNA damage comes in many forms that originate from intrinsic and extrinsic sources. These lesions can induce the mutations and genome rearrangements that lead to cancer, aging and degenerative diseases. Particularly pathological lesions are the DNA double stranded DNA breaks (DSBs), as well as collapsed replication forks caused by barriers to replication, that can themselves promote DSB formation. To activate cell cycle checkpoints, mechanisms that allow the time to repair these lesions, ssDNA is generated at these sites in a 5?3? direction. The resulting ssDNA that remains has an exposed 3?-OH group, and acts as a landing pad for assembly of checkpoint signaling complexes as well as recombination enzymes that promote invasion into the sister chromatid. Using the fission yeast Schizosaccharomyces pombe as a gene and pathway discovery tool, we identified a family of XPG-related nucleases (XRNs) as the long sought after enzymes that are necessary and sufficient for end resection at DSBs. This consists of the long known Rad2/Fen1 and Exo1 enzymes that also function in Okazaki Fragment maturation and various Excision Repair pathways. The newly identified third member of this family is known as the Asteroid nucleases. These include Ast1 in S. pombe and ASTE1 in humans, but there is no Asteroid homolog in the budding yeast Saccharomyces cerevisiae. Thus, Ast1 enzymes remain poorly characterized compared to Fen1 and Exo1. Studies leading to, and during the initial funding period of this grant, have shown that these XRN nucleases are hierarchically recruited to DSBs, which is dynamic depending on the complement of nucleases. There is further specificity afforded by the direction of transcription at a damaged locus. A considerable body of data also shows that the XRNs are critical at collapsed replication forks, cooperating with several other enzymes that modulate fork stability and processing. Additional experiments in this proposal build on these observations and utilize an armory of new tools to study the processing of these lesions across the genome. We present a thorough analysis of Ast1 to bring the understanding of this conserved enzyme to level commensurate with its long-studied cousins, then determine specificity determinants among them. As these initiating events in DNA damage responses feed into many downstream response pathways, this work has significant impact on the study of the many mechanisms that ensure the integrity of the genome.